CN111000101B - 一种具有高效解酒护肝的组合物及包含其的饮品 - Google Patents
一种具有高效解酒护肝的组合物及包含其的饮品 Download PDFInfo
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- CN111000101B CN111000101B CN201911115613.9A CN201911115613A CN111000101B CN 111000101 B CN111000101 B CN 111000101B CN 201911115613 A CN201911115613 A CN 201911115613A CN 111000101 B CN111000101 B CN 111000101B
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Abstract
本发明公开了一种具有高效促进乙醇代谢、护肝的组合物,所述组合物包含鼠李糖乳杆菌EH132菌株、葛根提取物和藤茶提取物。本发明的乙醇代谢、解酒护肝组合物中含有鼠李糖乳杆菌EH132菌株发酵上清液及中药成分葛根提取物和藤茶提取物,鼠李糖乳杆菌EH132菌株与葛根提取物和藤茶提取物可发挥协同作用,具有高效促进乙醇代谢、解酒护肝的功能,且所述组合物绿色无毒,符合食品安全规范,可用于进一步制备食品或保健品,具有较大的应用前景。
Description
技术领域
本发明涉及食品制剂的技术领域。更具体地,涉及一种具有高效解酒护肝的组合物及包含其的饮品。
背景技术
随着人们生活水平的不断提高,饮酒过度已经成为一个比较严重的公共卫生问题。我国因过度饮酒而造成肝脏损害的人数也在逐年增多。饮酒过多会使肝细胞发生细胞水肿,脂肪变性等病理改变。长期大量饮酒则会出现慢性酒精中毒,脂肪肝,肝炎,肝硬化,甚至最终转化成肝癌。近年来,饮酒过多造成的酒精性肝损伤越来越成为人们重点关注的问题。为了减少这种问题的产生,饮用解酒护肝的产品成为热点。能够高效促进酒精类产品的代谢且能护肝的产品具有较好的市场前景,对于由饮酒过度引发的肝脏疾病具有重要的预防和治疗作用。
目前市面上出现的解酒护肝产品包括中药解酒片,以葛根和葛花为主的植物酵素、保健品等,例如专利CN106035806A公开了一种茶粉组合物,包含茶粉1~50份,益生菌0.1~20份,功能糖1~30份以及第二成分30~50份;所述第二成分包括蒲公英粉、五味子粉、甘草粉、罗汉果粉、红枣粉、枸杞粉、葛根粉、枳椇子粉、玉米低聚肽和牛磺酸中的至少两种以上;专利CN109007808A公开了一种解酒护肝组合物,包含玉米低聚肽10~55份、姜黄2~20份、葛根提取物2~20份、枳椇子提取物5~20份、西兰花种子水提物0.5~23份;专利CN1454526A公开的一种帮助解酒的功能保健饮料,主要是采用藤茶、佛手、桔红、葛根、甘草经提取添加牛磺酸、浓缩鲜橙汁、柠檬汁和蜂蜜等组分精制而成。然而,上述具有解酒护肝功能的保健品,大多成分复杂,不仅增加了解酒产品的成本和不可控的成分,而且解酒酒护肝效果不够理想。
发明内容
本发明的目的在于克服现有技术中存在的上述缺陷和不足,提供一种具有高效解酒护肝的组合物。
本发明的另一目的在于提供一种包含所述解酒护肝组合物的饮品。
本发明的上述目的是通过以下技术方案给予实现的:
一种具有高效解酒护肝的组合物,包含鼠李糖乳杆菌EH132菌株、葛根提取物和藤茶提取物;所述鼠李糖乳杆菌EH132菌株于2019年09月02日保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC No.60757。
本发明的组合物中,包含鼠李糖乳杆菌EH132菌株与中药成分葛根提取物及藤茶提取物。本发明研究发现,鼠李糖乳杆菌EH132菌株具有良好的解救护肝功能,而且当鼠李糖乳杆菌EH132菌株益生菌与具有解酒功能的葛根提取物和藤茶提取物混合使用时具有较好的协同作用、更高效的发挥解酒护肝作用;与单独使用鼠李糖乳杆菌EH132菌株、葛根提取物或藤茶提取物相比,具有更好的解酒护肝效果。
优选地,所述组合物包含鼠李糖乳杆菌EH132菌株发酵上清液、葛根提取液和藤茶提取液。
优选地,所述葛根提取液浓度为5mg/mL,藤茶提取物浓度成1mg/mL。
优选地,所述组合物中鼠李糖乳杆菌EH132菌株发酵上清液、葛根提取液和藤茶提取液的体积比为(1~2):(1~3):(1~5)。
优选地,所述组合物中鼠李糖乳杆菌EH132菌株发酵上清液、葛根提取液和藤茶提取液的体积比为(1~3):1:1。
更优选地,所述组合物中鼠李糖乳杆菌EH132菌株发酵上清液、葛根提取液和藤茶提取液的体积比为1:1:1。
所述鼠李糖乳杆菌EH132菌株发酵液按照本领域常规的鼠李糖乳杆菌发酵液制备方法即可。优选地,所述鼠李糖乳杆菌EH132菌株发酵上清液的制备方法为先活化菌种,再将活化好的菌株以1%接种量接种至发酵培养基中,30~37℃(优选37℃)发酵70~80h(优选72h),调整发酵后菌液的OD至1.0~1.4(优选1.2),离心,取上清,即得鼠李糖乳杆菌EH132发酵上清液。
优选地,所述活化培养基为MRS固体培养基,发酵培养基为MRS肉汤培养基。
优选地,所述离心为4℃14000r/min离心15min。
优选地,所述葛根提取物的制备方法包括如下步骤:取新鲜葛根切成薄片,烘干,磨成粉末;取适量的葛根粉末,以70%乙醇为提取溶剂,提取固液比为1∶12~17(g/mL)(优选15g/mL),超声温度55~65℃(优选60℃),提取20~30min(优选25min);将提取液中的乙醇旋干后,制备成提取物粉末,避光干燥保存备用。
优选地,所述藤茶提取物的制备方法为:以热水为提取溶剂,热水与藤茶干叶的液料比为30:1~2,60~80℃,pH 7~8,提取50~70min;抽滤,滤液冷却结晶,再次抽滤后干燥,得到粗提物;将上述所得的粗提物纯化,用丙酮提取液回流提取两次,液料比分别为5:1和3:1,之后多次重结晶,反复4次,得到纯度较高的提取物晶体,干燥保存备用。
本发明还请求保护上述任一所述的组合物在制备具有高效促进乙醇代谢、解酒护肝制剂、食品或保健品中的应用。
一种具有高效解酒护肝的制剂,包含上述任一所述的组合物。
优选地,所述制剂为饮液型。
优选地,所述饮液型制剂的制备方法包括以下步骤:按照体积比量取各原料组分,将各原料组分溶解于80~100份水中,得到饮液型解酒护肝制剂。
与现有技术相比,本发明具有以下有益效果:
本发明的解酒护肝组合物中含有鼠李糖乳杆菌EH132菌株发酵上清液、葛根提取物和藤茶提取物,鼠李糖乳杆菌EH132菌株与葛根提取物和藤茶提取物中药成分可发挥协同作用,具有高效促进乙醇代谢、解酒护肝的功能,且所述组合物绿色无毒,符合食品安全规范,可用于进一步制备食品或保健品,具有较大的应用前景。
具体实施方式
以下结合具体实施例来进一步说明本发明,但实施例并不对本发明做任何形式的限定。除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明,以下实施例所用试剂和材料均为市购。
实施例1鼠李糖乳杆菌EH132菌株的分离培养
1、培养基配置
(1)MRS固体培养基:蛋白胨10g,牛肉膏粉5g,酵母膏粉4g,葡萄糖20g,吐温-801mL,磷酸氢二钾2g,乙酸钠5g,柠檬酸三铵2g,硫酸镁(MgSO4·7H2O)0.2g,硫酸锰(MnSO4·4H2O)0.05g,琼脂15g,加1L水,121℃高压灭菌15~20min。
(2)MRS肉汤培养基:蛋白胨10g,牛肉膏粉5g,酵母膏粉4g,葡萄糖20g,吐温-801mL,磷酸氢二钾2g,乙酸钠5g,柠檬酸三铵2g,硫酸镁(MgSO4·7H2O)0.2g,硫酸锰(MnSO4·4H2O)0.05g,加1L水,121℃高压灭菌15~20min。
2、鼠李糖乳杆菌EH132菌株的分离培养
(1)菌悬液的配制
先将人肠道粪便搅拌均匀,用无菌移液管吸取样品10mL加入盛有90mL无菌水的三角瓶中,在旋涡均匀器上充分振摇20分钟,使样品均匀分散。用一支1mL无菌吸管从中吸取1mL粪便菌液悬液注入9mL无菌水的试管中,吹息三次,使充分混匀。然后再用一支1mL无菌吸管从此试管中吸取1mL注入另一盛有9mL无菌水的试管中,以此类推制成10-1、10-2、10-3、10-4、10-5、10-6、10-7各种稀释度的菌悬液(要取7只15mL试管)。
(2)倒平板
取无菌平板12个,编号标明10-1、10-5、10-6、10-7各三套;将经121℃灭菌30min的MRS固体培养基冷却到65℃左右,按无菌操作法倒12只平板,每皿约15mL;平置使培养基均匀分布在皿底,待凝固。
3、分离方法
(1)划线在近火焰处,左手拿皿底,右手拿接种环,按无菌操作对平板进行划操作。
(2)用接种环以无菌操作挑取菌液一环,先在平板培养基的一边作第一次平行划线3~4条,再转动培养皿约70°角,并将接种环上剩余物烧掉,待冷却后通过第一次划线部分做第二次平行划线,再用同法通过第二次平行划线部分做第三次平行划线和通过第三次平行划线部分作第四次平行划线。划线完毕后,盖上皿盖,倒置于放在37℃恒温培养箱中培养48小时。将培养后长出的单个菌落分别挑取接种到新的MRS固体培养基上,查看是否纯培养,若不是,继续分离培养直至获得纯培养,得到了一个纯培养菌株EH132;革兰氏染色为阳性,无芽孢,短小杆状,不运动,最适生长温度为37℃。能发酵鼠李糖、核糖、山梨醇、乳糖、麦芽糖、松三糖、果糖、七叶灵和纤维二糖。提取菌株EH132的16S rRNA基因并测序,其序列如SEQ ID NO.1所示,将其与NCBI网站数据库进行BLAST分析,结果分析表明,其与菌株Lactobacillus rhamnosus的模式菌种的相似性最高,其中与LactobacillusrhamnosusDSM 20021T的序列相似性为99%以上。综合上述的生理生化特性、16S rRNA基因序列结果,鉴定菌株EH132属于Lactobacillus rhamnosus,将该菌株命名为鼠李糖乳杆菌EH132(Lactobacillus rhamnosus EH132),并将该菌于2019年09月02日保藏于广东省微生物菌种保藏中心(GDMCC),地址:广州市先烈中路100号大院59号楼5楼,保藏编号为GDMCCNO:60757。
实施例2
1、原料组分制备
(1)鼠李糖乳杆菌EH132菌株发酵上清液制备
采用MRS固体培养基活化菌种,37℃培养箱培养48h;之后将活化好的菌株取2环接种到MRS肉汤培养基(MRS固体培养基不加琼脂)中,37℃发酵72h。调整发酵后菌液的OD至1.2;14000r/min,4℃,离心15min,留取上清,待用。
(2)葛根提取物制备
取新鲜葛根切成薄片,60℃烘干。干燥后的葛根冷却至室温后,用粉碎机磨成粉末,4℃避光保存。取适量的葛根粉末,以70%乙醇为提取溶剂,提取固液比为1∶15(g/mL),超声温度60℃,提取时间25min。将提取液中的乙醇旋干后,制备成提取物粉末,避光干燥保存备用。
(3)藤茶提取物
以热水为提取溶剂,提取藤茶干叶中的有效成分,液料比为30:1。提取条件为:温度70℃,提取时间60min,pH 8.0。再经过抽滤,得到滤液后冷却结晶,再次抽滤后干燥,得到粗提物。将上述所得的粗提物纯化,用丙酮提取液回流提取两次,液料比分别为5:1和3:1,之后多次重结晶,反复4次,得到纯度较高的提取物晶体,干燥保存备用。
2、组合物制备
将葛根提取物配置成5mg/mL的葛根提取液;藤茶提取物配置成1mg/mL的藤茶提取液。按表1所示的组分和体积比分别量取步骤各组分,混匀,配置成10mL的溶液。
表1
试验例1
测定上述组1~组9所述组合物的体外乙醇脱氢酶性能
采用改良后的瓦勒-霍赫(Valle&Hoch)法,测定乙醇脱氢酶的活性。将1.5mL 32mM的焦磷酸钠缓冲液(PH8.8),1.0mL 27mM NAD+溶液,0.5mL 11.5%(v/v)乙醇,0.1mL组合物混匀,25℃温育5min后,立即加入0.1mLADH(0.64μg/mL)溶液。对照组用0.1mL蒸馏水代替样品溶液,其余操作相同立即在340nm波长下,每隔10s读数一次,连续测定5min。乙醇脱氢酶活性计算法以A对时间作图,计算A340/10s的增加值,根据NADH在340nm处的摩尔消光系数为6.22,计算酶活力。ADH的活力以每分钟NADH生成的纳摩尔数来表示。
式中:V为总反应液的体积(m L);DF为稀释倍数;6.22为NADH在340nm波长下的毫摩尔消光系数;0.1为酶液的体积(m L)。组1~组9所述组合物的乙醇脱氢酶能力如表2所示:
表2
试验例2
测定试验例1中有最大酶活的组合物,即组7,以及组1~3的体外乙醛脱氢酶性能。
采用改良的Blair&Bodley方法。在测定管中分别加入100mM pH值为9.5的焦磷酸钠缓冲液1.6mL、3.6mM氧化型辅酶I(NAD+)1m L,100mM乙醛溶液0.1m L,10mM吡唑0.1m L样品溶液0.1m L,混合后,放入30℃的水浴锅中,加盖温育5min。再向测定管中加入18mM ALDH0.1mL,摇匀后立即用分光光度计测定其吸光度(A340)值,以后每隔1min读数1次,直至每分钟吸光度的增大值达到稳定为止。以A值对时间作图,计算A340/1min的增加值,根据NADH在340nm处的摩尔吸光系数为6.22,计算酶活力单位。ALDH的活力以每分钟NADH生成的纳摩尔数来表示,计算公式如下:
式中:V为总反应液的体积(mL);DF为稀释倍数;6.22为NADH在340nm波长下的毫摩尔消光系数;0.1为酶液的体积(mL)。组1~3及组7所述组合物乙醛脱氢酶能力如表3所示:
表3
试验例3
测定实施组7和对比组1~3提供的饮液型制剂体外抗氧化能力实验
DPPH自由基广泛用于研究抗氧化物质的自由基清除活性的测定,测定方法参照Rathee等。用0.1mL的样品加入到试管中,然后向每管中加入3.9mL 0.1mmol/L DPPH乙醇溶液,混匀避光37℃水浴1h后在517nm下测定吸光度值,以乙醇做空白对照,每个样品均平行测定三次,结果以SC50值表示。DPPH自由基清除率为50%时所需样品的浓度为SC50,DPPH自由基清除率按下式计算:
清除率(%)=[1-(As/Ac)]x100%
式中:Ac——空白对照管的吸光度值;As——样品测定管的吸光度值。
组1~3及组7所述组合物体的外抗氧化能力如表4所示:
表4
实施例3
按照实施例2表1中组4~9所示组合物配比,配置3种葛根提取物和藤茶提取物的混合溶液;3种鼠李糖乳杆菌EH132菌株发酵上清液、葛根提取物和藤茶提取物的混合溶液,再分别取1份混合液,分别溶解于100份水中,得到饮液型制剂。
实施例4
按照实施例2表1中组4~9所示组合物配比,配置3种葛根提取物和藤茶提取物的混合溶液,3种鼠李糖乳杆菌EH132菌株发酵上清液、葛根提取物和藤茶提取物的混合溶液,再分别取1份混合液,分别溶解于90份水中,得到饮液型制剂。
实施例5
按照实施例2表1中组4~9所示组合物配比,配置3种葛根提取物和藤茶提取物的混合溶液,3种鼠李糖乳杆菌EH132菌株发酵上清液、葛根提取物和藤茶提取物的混合溶液,再分别取1份混合液,分别溶解于80份水中,得到饮液型制剂。
本发明上述结果表明,葛根提取物和藤茶提取物组合使用较葛根提取物胡藤茶提取物单独使用具有更显著的解救护肝功能,尤其是当鼠李糖乳杆菌EH132与葛根提取物和藤茶提取物混合使用时,鼠李糖乳杆菌EH132菌株发酵上清液可以与葛根提取物和藤茶提取物中药成分可发挥协同作用,具有高效促进乙醇代谢、解酒护肝的功能。
序列表
<110> 南方医科大学
<120> 一种具有高效解酒护肝的组合物及包含其的饮品
<141> 2019-11-14
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1449
<212> DNA
<213> 鼠李糖乳杆菌EH132(Lactobacillus rhamnosus EH132)
<400> 1
cttagacggc tcgctcccta aaagggttac gccaccggct tcgggtgtta caaactctca 60
tggtgtgacg ggcggtgtgt acaaggcccg ggaacgtatt caccgcggcg tgctgatccg 120
cgattactag cgattccgac ttcgtgtagg cgagttgcag cctacagtcc gaactgagaa 180
tggctttaag agattagctt gacctcgcgg tctcgcaact cgttgtacca tccattgtag 240
cacgtgtgta gcccaggtca taaggggcat gatgatttga cgtcatcccc accttcctcc 300
ggtttgtcac cggcagtctt actagagtgc ccaactaaat gctggcaact agtcataagg 360
gttgcgctcg ttgcgggact taacccaaca tctcacgaca cgagctgacg acaaccatgc 420
accacctgtc attttgcccc cgaaggggaa acctgatctc tcaggtgatc aaaagatgtc 480
aagacctggt aaggttcttc gcgttgcttc gaattaaacc acatgctcca ccgcttgtgc 540
gggcccccgt caattccttt gagtttcaac cttgcggtcg tactccccag gcggaatgct 600
taatgcgtta gctgcggcac tgaagggcgg aaaccctcca acacctagca ttcatcgttt 660
acggcatgga ctaccagggt atctaatcct gttcgctacc catgctttcg agcctcagcg 720
tcagttacag accagacagc cgccttcgcc actggtgttc ttccatatat ctacgcattt 780
caccgctaca catggagttc cactgtcctc ttctgcactc aagtttccca gtttccgatg 840
cacttcctcg gttaagccga gggctttcac atcagactta aaaaaccgcc tgcgctcgct 900
ttacgcccaa taaatccgga taacgcttgc cacctacgta ttaccgcggc tgctggcacg 960
tagttagccg tggctttctg gttggatacc gtcacgccga caacagttac tctgccgacc 1020
attcttctcc aacaacagag ttttacgacc cgaaagcctt cttcactcac gcggcgttgc 1080
tccatcagac ttgcgtccat tgtggaagat tccctactgc tgcctcccgt aggagtttgg 1140
gccgtgtctc agtcccaatg tggccgatca acctctcagt tcggctacgt atcattgcct 1200
tggtgagccg ttacctcacc aactagctaa tacgccgcgg gtccatccaa aagcgatagc 1260
ttacgccatc tttcagccaa gaaccatgcg gttcttggat ttatgcggta ttagcatctg 1320
tttccaaatg ttatccccca cttaagggca ggttacccac gtgttactca cccgtccgcc 1380
actcgttcaa aattaaatca agatgcaagc acctttcaat aatcagaact cgtcgactgc 1440
attatagca 1449
Claims (8)
1.一种具有高效促进乙醇代谢、护肝的组合物,其特征在于,包含鼠李糖乳杆菌EH132菌株发酵上清液、葛根乙醇提取液和藤茶水提取液;所述鼠李糖乳杆菌EH132菌株于2019年09月02日保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC No.60757。
2.根据权利要求1所述的组合物,其特征在于,所述鼠李糖乳杆菌EH132菌株发酵上清液、葛根乙醇提取液和藤茶水提取液的体积比为(1~2):(1~3):(1~5)。
3.根据权利要求2所述的组合物,其特征在于,所述鼠李糖乳杆菌EH132菌株发酵上清液、葛根乙醇提取液和藤茶水提取液的体积比为1:1:1。
4.根据权利要求1所述的组合物,其特征在于,所述鼠李糖乳杆菌EH132菌株发酵上清液的制备方法为先活化菌种,再将活化好的菌株以1%接种量接种至发酵培养基中,30~37℃发酵70~80h,调整发酵后菌液的OD至1.0~1.4,离心,取上清,即得鼠李糖乳杆菌EH132发酵上清液。
5.权利要求1~4任一所述的组合物在制备解酒护肝制剂、食品或保健品中的应用。
6.一种具有高效解酒护肝的制剂,其特征在于,包含权利要求1~4任一所述的组合物。
7.根据权利要求6所述的制剂,其特征在于,所述制剂为饮液型。
8.根据权利要求7所述的制剂,其特征在于,所述制剂的制备方法包括以下步骤:按照体积比量取各原料组分,将各原料组分溶解于80~100份水中,得到饮液型解酒护肝制剂。
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